High-quality genomic DNA was extracted from the untreated PSC-1 strain and from the treated samples at each different SbIII concentration (50, 100, 200, 400, 800, 1600, and 2000 µM SbIII), as well as from the two clinical isolates. Genomic DNA was extracted from stationary phase promastigotes, using a commercial salting out procedure as recommended by the manufacturer (Wizard Genomic DNA purification kit, Promega, Madison, WI, USA). Size, integrity, and presence of contaminants in the DNA samples were assessed through gel electrophoresis. DNA concentration was estimated by the picogreen method (Invitrogen, Carlsbad, CA, USA) using Victor 3 fluorometry (PerkinElmer, Waltham, MA, USA). DNA purity was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Sequencing libraries with 150 bp shotgun (paired-end) reads were prepared from 100 ng of genomic DNA by using the TruSeq Nano sample preparation protocol (Illumina, Inc., San Diego, CA, USA) and then sequenced in a NovaSeq 6000 instrument for a total throughput of 4 Gb per sample.
Picogreen method
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Picogreen method is a fluorescence-based technique for quantifying double-stranded DNA (dsDNA) in solution. It utilizes a fluorescent dye that binds specifically to dsDNA, allowing for the measurement of DNA concentration with high sensitivity. The Picogreen method provides a simple and reliable way to determine the amount of dsDNA present in a sample.
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Lab products found in correlation
2100 bioanalyzer, by Illumina (2 mentions)
Chip seq protocol chip seq sample preparation kit, by Illumina (2 mentions)
Genome analyzer, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Victor 3 fluorometry, by PerkinElmer (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Faststart high fidelity pcr system, by Roche (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Gs flx titanium sequencer, by Roche (1 mentions)
Ht7900 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iplex primer extension, by Labcorp (1 mentions)
Repli g mini kit, by Qiagen (1 mentions)
Powerwater rna isolation kit, by Qiagen (1 mentions)
Epitect fast dna bisulfite kit, by Qiagen (1 mentions)
Ez1 dna investigator kit, by Qiagen (1 mentions)
12 protocols using picogreen method
1
Genomic DNA Extraction and Sequencing
2
16S rRNA Amplification and Sequencing
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After each clone was polymerase chain reaction (PCR)-amplified, the V1 to V3 regions were amplified using a FastStart High Fidelity PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with a 16s rRNA gene fusion primer (27F-GAGTTTGATCMTGGCTCAG and 518R-WTTACCGCGGCTGCTGG). A GS-FLX plus emPCR Kit (454 Life Sciences, Branford, CT, USA) and Tissue Lyser II (Qiagen, Germantown, MD, USA) were used to make micro-reactors containing amplified compounds and beads. After emPCR amplification, the amplicon was purified using an AMpure Bead kit (Beckman Coulter, Brea, CA, USA) and quantified using the Picogreen method (Invitrogen, Carlsbad, CA, USA). Subsequently, the amplicon was diluted and analyzed with a GS-FLX Titanium sequencer (Roche Diagnostics GmbH) at Macrogen (Seoul, Korea).
3
Hippocampal Gene Expression in HIV-1 Tg Rats
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A separate cohort of rats raised under the same conditions (WT n = 10; HIV-1 tg n = 9), was used for gene expression analysis. Animals were rapidly decapitated, brain tissue was dissected frozen under RNAse-free conditions, and the hippocampus was isolated for analysis. Samples were then homogenized using the Qiagen RNeasy Mini Kit (Valencia, CA) according to manufacturer’s instructions. RNA samples were reverse transcribed using Applied Biosystem’s High Capacity cDNA Reverse Transcription Kit. Resulting cDNA was then quantified and normalized using the PicoGreen method (Invitrogen, Grand Island, NY). The TaqMan gene expression system was used for the detection of monocyte chemotactic protein-1 (Mcp-1; Rn00580555_m1), interleukin-1β (Il-1β; Rn00580432_m1), nuclear factor-kappa-β inhibitor, α (Nf-κBiα; RN01473654_g1), tumor necrosis factor (Tnf; Rn00562055_m1), and standardized to β-actin (Rn00667869_m1). All samples were prepared in triplicate using 1 µg of sample and carried out on an Applied Biosystems HT7900 Fast Real-Time PCR system. Data were averaged by group and analyzed via the ΔΔCt method.
4
ChIP-seq Library Preparation Protocol
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After crosslink reversion, phenol-chloroform extraction and ethanol precipitation, the DNA from remodeller-nucleosome complexes was quantified using the picogreen method (Invitrogen) or by running 1/20 of the ChIP material on a High sensitivity DNA chip on a 2100 Bioanalyzer (Agilent, USA). 5 to 10 ng of ChIP DNA were used for library preparation according to the Illumina ChIP-seq protocol (ChIP-seq sample preparation kit). Following end-repair and adapter ligation, fragments were size-selected on an agarose gel in order to purify nucleosome-sized genomic DNA fragments between 140 and 180 bp. Purified fragments were next amplified (18 cycles) and verified on a 2100 Bioanalyzer before clustering and single-read sequencing on an Illumina Genome Analyzer (GA) or GA II, according to manufacturer’s instructions. Sequencing characteristics are shown in Supplementary Table 1 .
5
ChIP-seq Library Preparation Protocol
6
Genetic Variant Profiling of Immunoglobulin Loci
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Genomic DNA was extracted from whole blood using the Chemagen Magnetic Bead Technology (Chemagic MSM I, Baesweiler, Germany). DNA preparations were quantified using the PicoGreen method (Invitrogen/Life Technologies, Paisley, UK) according to supplier instructions. SNP selection criteria took into account allelic frequency in the Caucasian populations, available sequencing confirmation, and tagging of LD blocks (http://www.hapmap.org ). The SNP genotyping method used the Mass Array system to design multiplex reactions for PCR, iPlex primer extension (Sequenom, San Diego, CA, USA) and the MALDI-TOF-based Mass Array platform (Sequenom). A total of 239 patients, 169 non-affected parents, and 130 unrelated healthy controls were genotyped for 15 SNPs mapping in 4 regions in immunoglobulin heavy chain locus: 4 SNPs in the IgHG, 4 in IgHD, 5 in IgHM, and 2 in IgHV regions. Genotyping quality control selected 9 SNPs that yield correct genotyping data according to HapMap control samples and passed the Hardy-Weinberg equilibrium test (P > 0.05) with a call rate above 80%.
7
DNA Extraction from Guthrie Cards
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DNA was extracted from archived Guthrie cards obtained from the 389 subjects. Two 3-mm punches from each card were incubated with the GenSolve reagent (IntegenX, Pleasanton, CA) for 1 h in a shaking heat block at 65°C. After centrifugation to remove the paper, elute was purified using the QIAamp DNA Mini Kit (Cat. No. 51306; Qiagen, Germantown, MD) according to the manufacturer's instructions, with the modification that the elution step was performed with preheated buffer at 70°C, followed by incubation of the spin columns at 70°C for 10 min. DNA was recovered by centrifugation. DNA concentrations were measured using the PicoGreen method (Life Technologies, Carlsbad, CA).
Genomic DNA was directly used for MLPA experiments. Whole genome amplified (WGA) DNA was used for array-CGH, Single-nucleotide polymorphism (SNP) genotyping, PCR breakpoint junction cloning experiments, and sequencing. Genomic DNA was amplified using GenomePlex kit (Sigma, Saint Louis, MO) and purified using Sephadex G50 spin column for array-CGH and PCR breakpoint junction cloning experiments. Similarly, we amplified genomic DNA using REPLI-g Mini Kit (QIAGEN) for SNP genotyping and DNA sequencing.
Genomic DNA was directly used for MLPA experiments. Whole genome amplified (WGA) DNA was used for array-CGH, Single-nucleotide polymorphism (SNP) genotyping, PCR breakpoint junction cloning experiments, and sequencing. Genomic DNA was amplified using GenomePlex kit (Sigma, Saint Louis, MO) and purified using Sephadex G50 spin column for array-CGH and PCR breakpoint junction cloning experiments. Similarly, we amplified genomic DNA using REPLI-g Mini Kit (QIAGEN) for SNP genotyping and DNA sequencing.
8
Nucleic Acid Extraction from Sea Ice and Seawater
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DNA extractions were performed on duplicate samples of the melted sea ice (50–100 ml) or seawater samples (1000 ml) filtered on 0.22 μm nitrocellulose filters (Millipore, Billerica, MA, USA). The filters were immediately frozen and stored at −80 °C until DNA and RNA extraction. The Powerwater RNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) was used for seawater nucleic acid extraction of the filters according to the manufacturer’s protocol, modified for the presence of lysis-resistant organisms and the omission of DNAse treatment for total nucleic acid extraction. The protocol for DNA extraction of the sea ice filters was based on a modified CTAB (cetyltrimethylammonium bromide) method of Ausubel et al.17 to address the large amounts of exopolymeric substances found on the sea ice filters. Volumes of reagents were tripled in comparison to the original protocol to ensure full submersion of filters. DNA was quantified by the Picogreen method (Life Technologies, Burlington, On, Canada) on a Tecan Magellan Fluorimeter (Tecan Group Ltd., Männedorf, Switzerland). DNA extracts were stored at −80 °C until further analysis.
9
DNA Extraction and Modification Protocol
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In total, 10 blood samples, 9 buccal swabs, and 7 semen samples were collected from volunteers according to the approved IRB-13-0555 from Florida International University. Swabs were air-dried, and DNA extraction was performed using the EZ1 DNA Investigator Kit (Qiagen, Valencia, CA, USA) and the BioRobot EZ1 automated purification workstation (Qiagen) according to the manufacturer's specifications. Quantification was performed using the PicoGreen method (Life Technologies, Carlsbad, CA, USA). DNA (1 and 50 ng) was bisulfite modified using the EpiTect Fast DNA Bisulfite Kit (Qiagen) according to the manufacturer's instructions.
For the sensitivity studies, samples were serial diluted to obtain 0.5 and 0.25 ng of input DNA to bisulfite conversion. In parallel, the DNA samples that were modified using 1 ng of input to bisulfite modification were serially diluted after modification to determine the lowest amount amplifiable by HRM. Samples that were not bisulfite modified were diluted to 0.5 ng/ml in order to obtain a 1 ng input to the HRM reaction.
For the sensitivity studies, samples were serial diluted to obtain 0.5 and 0.25 ng of input DNA to bisulfite conversion. In parallel, the DNA samples that were modified using 1 ng of input to bisulfite modification were serially diluted after modification to determine the lowest amount amplifiable by HRM. Samples that were not bisulfite modified were diluted to 0.5 ng/ml in order to obtain a 1 ng input to the HRM reaction.
10
Biliary Duct Cells Microbiome Analysis
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Epithelial cells were scraped from the biliary duct with brushes (Cytomax II; Cook Medical Inc., Bloomington, IN, USA) during ERCP and maintained at -80°C until processed. Brushings were done in the common bile duct, in the proximity of the gallbladder or in the region corresponding to the ampulla of Vater, depending on the site of the lesion. DNA was extracted using the QIAamp DNA easy kit (Qiagen, Hilden, Germany), and quantified by PicoGreen method (Life Technologies, Carlsbad, CA, USA). Helicobacter bilis and H. hepaticus were investigated by PCR in all 100 ECCA and 100 BBP patients, as reported previously [6] , and 20 patients were selected for microbiota studies: five Helicobacter-positive cases and five controls, and five Helicobacter-negative cases and five controls (Table 1).
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